The Journal of Experimental Medicine
JEM © The Rockefeller University Press $30.00
Vol. 204, No. 10, October 1, 2007 2349-2362 www.jem.org/cgi/doi/
Lymphatic vessels are key routes for the recir-
culation of fl uid and cells that enter tissues from
blood vessels. This function of lymphatics is
important for maintenance of normal tissue
homeostasis and in infl ammatory diseases and
other conditions with extensive fluid and
cell effl ux ( 1 ). Lymphatics are also routes for
spreading cancer cells ( 1-3 ) and for antigen-
presenting cells traffi cking from tissues to
lymph nodes in immune surveillance ( 4, 5 ).
Imbalances in effl ux and recirculation of fl uid
or cells can result in lymphedema or disturbed
Fluid entry into lymphatics is driven largely
by hydrostatic and colloidal osmotic pressure
gradients ( 6, 7 ). A prevailing view is that much
of the endothelium of initial lymphatics has in-
complete or no intercellular junctions ( 8-10 ).
The loosely apposed but overlapping borders
of endothelial cells are thought to function as
“ primary valves ” that provide unidirectional
fl uid fl ux into lymphatics ( 11, 12 ). When lym-
phatic endothelial cells are pulled apart by an-
choring fi laments tensioned by interstitial forces,
lymph fl ows along its pressure gradient into
lymphatics ( 8, 10, 11 ).
The properties of leukocyte entry into lym-
phatics diff er from those for fl uid, as leukocyte
infl ux is a selective process. Dendritic cells,
macrophages, and lymphocytes enter lymphat-
ics, but neutrophils and erythrocytes generally
do not. Leukocytes are attracted by chemokines
from lymphatic endothelial cells and interact
Donald M. McDonald:
Abbreviations used: button,
button-like junction; EM,
electron microscopy; ESAM,
endothelial cell – selective adhesion
molecule; JAM-A, junctional
adhesion molecule – A; LYVE-1,
lymphatic vascular endothelial
hyaluronan receptor – 1; MHC
II, MHC class II; PECAM-1,
molecule – 1; VE-cadherin,
vascular endothelial cadherin;
zipper, zipper-like junction;
ZO-1, zonula occludens – 1.
P. Baluk and J. Fuxe contributed equally to this work.
The online version of this article contains supplemental material.
Functionally specialized junctions between
endothelial cells of lymphatic vessels
Peter Baluk, 1,2,3 Jonas Fuxe, 1,2,3 Hiroya Hashizume, 1,2,3 Talia Romano, 1,2,3
Erin Lashnits, 1,2,3 Stefan Butz, 4 Dietmar Vestweber, 4 Monica Corada, 5
Cinzia Molendini, 5 Elisabetta Dejana, 5,6,7 and Donald M. McDonald 1,2,3
1 Cardiovascular Research Institute, 2 Comprehensive Cancer Center, and 3 Department of Anatomy, University of California,
San Francisco, San Francisco, CA 94143
4 Max-Planck-Institute for Molecular Biomedicine and Institute of Cell Biology, University of M ü nster,
48149 M ü nster, Germany
5 FIRC Institute of Molecular Oncology Foundation, 20139 Milan, Italy
6 Department of Biomolecular Sciences and Biotechnology, Faculty of Sciences, University of Milan, 20133 Milan, Italy
7 Mario Negri Institute for Pharmacological Research, 20156 Milan, Italy
Recirculation of fl uid and cells through lymphatic vessels plays a key role in normal tissue
homeostasis, infl ammatory diseases, and cancer. Despite recent advances in understanding
lymphatic function (Alitalo, K., T. Tammela, and T.V. Petrova. 2005. Nature . 438:946–953),
the cellular features responsible for entry of fl uid and cells into lymphatics are incom-
pletely understood. We report the presence of novel junctions between endothelial cells of
initial lymphatics at likely sites of fl uid entry. Overlapping fl aps at borders of oak leaf – shaped
endothelial cells of initial lymphatics lacked junctions at the tip but were anchored on the
sides by discontinuous button-like junctions (buttons) that differed from conventional,
continuous, zipper-like junctions (zippers) in collecting lymphatics and blood vessels. How-
ever, both buttons and zippers were composed of vascular endothelial cadherin (VE-cadherin)
and tight junction – associated proteins, including occludin, claudin-5, zonula occludens – 1,
junctional adhesion molecule – A, and endothelial cell – selective adhesion molecule.
In C57BL/6 mice, VE-cadherin was required for maintenance of junctional integrity,
but platelet/endothelial cell adhesion molecule – 1 was not. Growing tips of lymphatic
sprouts had zippers, not buttons, suggesting that buttons are specialized junctions rather
than immature ones. Our fi ndings suggest that fl uid enters throughout initial lymphatics
via openings between buttons, which open and close without disrupting junctional integrity,
but most leukocytes enter the proximal half of initial lymphatics.
BUTTON-LIKE JUNCTIONS OF INITIAL LYMPHATICS | Baluk et al.
plexus of blood vessels ( Fig. 1 B ) and were two or three times
the size of mucosal venules ( Table I ). All lymphatic vessels in
the trachea expressed lymphatic vascular endothelial hyaluro-
nan receptor – 1 (LYVE-1) and Prox1, making positive identi-
fi cation straightforward despite the abundance of blood vessels
nearby ( Fig. 1, A – C ). Smooth muscle and luminal valves were
restricted to collecting lymphatics on the adventitial surface.
Buttons between endothelial cells of initial lymphatics
Immunohistochemical staining for VE-cadherin revealed
conspicuous diff erences between initial lymphatics and col-
lecting lymphatics in the trachea. Endothelial cells of initial
lymphatics were joined by discontinuous buttons ( Fig. 1 C ),
whereas endothelial cells of collecting lymphatics were joined
by continuous zippers ( Fig. 1 D ), similar to those in adjacent
blood vessels. Endothelial cells of lymphatics were apprecia-
bly larger than those in blood vessels ( Table I ).
Buttons consisted of roughly parallel linear segments of
VE-cadherin, 3.2 ? 0.1 ? m in length and spaced 2.9 ? 0.3 ? m
apart. Buttons were most abundant in the fi rst 500 ? m of
tracheal lymphatics and were rare beyond 1,500 ? m from the
tip ( Fig. 1 E ). The length of button-rich regions varied from
vessel to vessel, and scattered endothelial cells of some lym-
phatics had zippers, but the transition from buttons to zippers
was typically abrupt in individual vessels. Lymphatics in other
organs, including the diaphragm, urinary bladder, and skin
of ear and tail, had similar buttons in initial lymphatics and
zippers in collecting lymphatics (Fig. S1, available at http://
The unique association of buttons with initial lymphatics
was confi rmed by examining the distribution of VE-cadherin
in the context of LYVE-1. Double staining revealed that
buttons were at the perimeter of LYVE-1 – positive endothe-
lial cells with a distinctive oak leaf shape ( Fig. 1 F ). Cells of
this shape are typical of initial lymphatics and conspicuously
diff erent from spindle-shaped endothelial cells of collecting
lymphatics and blood vessels ( 9, 20 ). In initial lymphatics,
LYVE-1 was concentrated at the tip of scalloped edges (fl aps)
of the oak leaf – shaped cells, and segments of VE-cadherin
were located along the sides of fl aps ( Fig. 1 F ). The presence
with complementary adhesion molecules that govern ad-
hesion and migration ( 4, 13, 14 ). Yet, the specifi c routes cells
use to cross the lymphatic endothelium are at an early stage
Evidence that lymphatic endothelial cells make junctional
proteins comes from gene profi ling data, which document
the expression of platelet/endothelial cell adhesion molecule – 1
(PECAM-1; also known as CD31), junctional adhesion
molecule – A (JAM-A), and occludin in cultured cells ( 14-18 ).
Multiple adhesion molecules have also been reported at inter-
cellular junctions in specialized “ retothelial ” cells of lymph
node sinuses ( 19 ).
Among the unresolved questions about the entry of fl uid
and cells into lymphatics are the following: (a) how can the
integrity of initial lymphatics be maintained if junctions are
not present between endothelial cells; (b) if junctions are pre-
sent, how does fl uid enter without repetitive disruption of the
junctions; (c) do leukocytes and fl uid enter at the same sites;
and (d) what is the relation of the distinctive oak leaf shape of
endothelial cells of initial lymphatics ( 9, 20 ) to sites of fl uid
and cell entry?
Based on this background, we sought to learn whether the
properties of fl uid and cell entry into initial lymphatics could
be explained by the specialization of junctions between endo-
thelial cells instead of the absence of junctions. We compared
the distribution and composition of junctional proteins in
initial lymphatics to those of conventional intercellular junc-
tions in collecting lymphatics and blood vessels. Of particular
interest were vascular endothelial cadherin (VE-cadherin) of
adherens junctions, tight junction proteins, and the endothe-
lial adhesion molecule PECAM-1. After learning that initial
lymphatics had unusual, discontinuous endothelial junctions,
we tested junctional integrity and plasticity after inhibition of
VE-cadherin, deletion of PECAM-1, or increased fl uid and
cell fl ux in infl ammation.
The studies exploited the attributes of the mouse tracheal
mucosa, where both lymphatics and blood vessels are abun-
dant, easily visualized, and readily compared under baseline
conditions or after infl ammatory stimuli in wild-type or ge-
netically altered mice ( 21 ). High resolution confocal micro-
scopic imaging of three-dimensional whole mounts revealed
discontinuous, button-like junctions (buttons) in the endo-
thelium of initial lymphatics that contained proteins typical of
both adherens junctions and tight junctions but were struc-
turally unlike the zipper-like junctions (zippers) elsewhere.
These fi ndings suggest that regions between buttons in initial
lymphatics are openings where fl uid can enter without repet-
itive formation and dissolution of intercellular junctions.
Initial lymphatics of mouse trachea
In the mouse trachea, lymphatics, with rounded blind tips and
simple branching, were located in the mucosa between car-
tilage rings ( Fig. 1 A ). On average, 10.8 ? 0.4 blind ends of
lymphatics were present in each mucosal segment between car-
tilages. Lymphatic vessels were located beneath the subepithelial
Table I. Dimensions of endothelial cells of mouse lymphatics
and blood vessels
Vessel diameter ( ? m) 56 ? 1.5 (baseline)
68 ? 1.3 (LPS)
49 ? 1
18 ? 0.8
616 ? 40
64 ? 7 22 ? 1**
Cell length ( ? m)
Cell width ( ? m)
Cell area ( ? m 2 )
66 ? 3*
14 ? 0.6*
630 ? 40
40 ? 3**
11 ? 0.6**
312 ? 19**
Measured in mouse tracheal whole mounts stained immunohistochemically for
CD31 and LYVE-1 or VE-cadherin. All measurements were made under baseline
conditions except for diameter of initial lymphatics, which were prepared under
baseline conditions or 24 h after LPS. *, P ? 0.05 compared with initial lymphatics;
**, P ? 0.05 compared with initial lymphatics and collecting lymphatics.
JEM VOL. 204, October 1, 2007
Figure 1. Buttons in endothelium of initial lymphatics. (A) Confocal images showing lymphatic vessels (green, LYVE-1) and blood vessels
(red, PECAM-1) in whole mount of mouse trachea. Region of mucosa over horizontal cartilage (*) is mostly free of lymphatics. (B) Longitudinal section
of trachea shows epithelium (green), subepithelial blood vessels (red, arrowheads), more deeply positioned initial lymphatics (diagonal arrows), collecting
lymphatic (horizontal arrow), and adjacent cartilages. (C and D) Confocal images of VE-cadherin immunoreactivity (red) at discontinuous buttons in initial
lymphatic (arrows; C) and continuous zippers in collecting lymphatic (D). Zippers are also present in blood capillary (arrowheads; C). Lymphatics are iden-
tifi ed by Prox1 (green) in nuclei. (E) Distribution of 3,110 buttons along the length of 25 lymphatics in fi ve tracheas, expressed as a function of distance
from the tip. Values are presented as means ? SEM. *, P ? 0.05 compared with the number at the tip (0 ? m). (F and G) Confocal images showing VE-
cadherin at buttons (arrows) and LYVE-1 between buttons (arrowhead) at the border of oak leaf – shaped endothelial cells of initial lymphatic. (G) Enlarged
isosurface rendering of confocal image stack of boxed region in F. (H) Scanning electron microscopic image showing external surface of overlapping fl aps
at the junction of three endothelial cells of initial lymphatic. (I) Drawing of boxed region in H showing contributions of three endothelial cells. Bars:
(A and B) 100 ? m; (C, D, and F) 10 ? m; (G) 5 ? m; (H) 1 ? m.
BUTTON-LIKE JUNCTIONS OF INITIAL LYMPHATICS | Baluk et al.
proteins infl uenced the organization of buttons. To address this
issue, we asked whether loss or inactivation of either protein af-
fected the distribution of the other protein or the organization
of buttons. The eff ect of PECAM-1 deletion was examined in
PECAM-1 – null mice, which have no apparent vascular defects
under baseline conditions ( 27 ). In tracheas of PECAM-1 – null
mice, PECAM-1 immunoreactivity was absent in blood ves-
sels and lymphatics as expected, but VE-cadherin had a normal
button-like pattern in initial lymphatics and a normal zipper-
like pattern in blood vessels ( Fig. 4, A – C ). The pattern of VE-
cadherin at buttons in PECAM-1 – null mice ( Fig. 4 D ) was not
noticeably diff erent from wild-type mice ( Fig. 4 E ).
Alternatively, a function-blocking antibody (BV13) was
used to inactivate VE-cadherin ( 28 ). Genetic deletion of
VE-cadherin leads to embryonic lethality because adherens
junctions are essential for early steps of assembly of endothe-
lial cells into blood vessels ( 29, 30 ). VE-cadherin is similarly
important in the adult. The BV13 antibody causes disper-
sion of VE-cadherin away from junctions between endothe-
lial cells of blood vessels accompanied by fatal leakage and
hemorrhage within hours of intravascular injection ( 28 ). In
our experiments, injection of BV13 antibody, unlike normal
IgG of the same isotype ( Fig. 4 F ), led to dispersion of VE-
cadherin in endothelial cells of initial lymphatics and blood
vessels ( Fig. 4 G ). Surprisingly, BV13 also caused dispersion
of PECAM-1 in lymphatics and blood vessels ( Fig. 4 G ) but
did not alter the distribution of ZO-1 at buttons in initial
lymphatics ( Fig. 4, H and I ).
Zippers at the tips of lymphatic sprouts
We next addressed the question of whether initial lymphatics
are less diff erentiated regions of the lymphatic vasculature, and
whether buttons represent immature versions of zippers. In this
instance, we used a model of lymphatic sprouting, triggered
by inoculation of Mycoplasma pulmonis into the airways of mice
( 21 ), to determine whether buttons predominate at the tip of
lymphatic sprouts, as would be expected if the junctional
immaturity hypothesis is valid. At 14 d after infection, new
lymphatics were abundant in regions of mucosa over cartilage
rings where lymphatics were normally absent ( Fig. 5, A and B ).
The growing tips of the new lymphatics had zippers ( Fig. 5,
A and B , arrows) similar in appearance to those in collecting
lymphatics under baseline conditions ( Fig. 1 D ). Most regions
of lymphatics distal to the tip had oak leaf – shaped endothelial
cells with buttons at the perimeter ( Fig. 5 B , arrowheads). At
7 wk after infection, sprouts were less numerous, and most
lymphatics had buttons ( Fig. 5, C and D ) resembling those in
initial lymphatics under baseline conditions ( Fig. 1 C ).
Sites of leukocyte entry into lymphatics
The issue of whether button-rich regions of lymphatics are
preferential sites for leukocyte entry was explored by deter-
mining whether sites of cell migration coincided with but-
tons in a model of airway infl ammation. The relationship
of MHC class II (MHC II) – positive cells (mainly dendritic
cells and macrophages) to lymphatics was examined 24 h after
of VE-cadherin at the sides of fl aps explained the illusion that
buttons were oriented perpendicular to the cell border ( Fig.
1 C ). The complementary distribution of VE-cadherin and
LYVE-1 was particularly conspicuous after three-dimensional
isosurface rendering of confocal image stacks ( Fig. 1 G ).
Scanning electron microscopic examination of the exter-
nal surface of the endothelium of initial lymphatics, after ex-
posure of the plasma membrane by alkaline hydrolysis of
extracellular matrix ( 22, 23 ), revealed detailed features of the
interdigitating borders of adjacent oak leaf – shaped endothe-
lial cells ( Fig. 1 H ). Flaps at the scalloped borders of adjacent
endothelial cells interdigitated with one another and over-
lapped loosely ( Fig. 1, H and I ). These features were not pre-
sent at the continuous seams of adjacent endothelial cells of
collecting lymphatics (Fig. S1) or in blood vessels.
Junctional proteins at buttons
The composition of buttons in initial lymphatics was deter-
mined by comparing the extent of colocalization of VE-
cadherin and tight junction proteins. For the purposes of this
study, colocalization of two junction-associated proteins was
defi ned as pixels with fl uorescence signals in two separate
channels, rather than precise molecular colocalization within
the junction ( 24 ). VE-cadherin at buttons colocalized with
the tight junction protein occludin ( Fig. 2 A ). VE-cadherin at
buttons also largely matched the distributions of the classical
tight junction protein claudin-5 ( Fig. 2 B ), intracellular tight
junction protein zonula occludens – 1 (ZO-1; Fig. 2 C ), and
the recently identifi ed tight junction – associated Ig-like trans-
membrane proteins endothelial cell – selective adhesion mole-
cule (ESAM; Fig. 2 D ) ( 25 ) and JAM-A ( Fig. 2 E ) ( 26 ).
Despite striking structural diff erences in buttons and zippers,
the two types of junctions contained the same proteins. Oc-
cludin ( Fig. 2 F ) and claudin-5 (Fig. S1) were continuously
distributed at zippers in collecting lymphatics, where they
partially colocalized with VE-cadherin.
Relation of PECAM-1 to VE-cadherin at buttons
Endothelial cells of initial lymphatics and collecting lymphatics,
like those in blood vessels, had PECAM-1 immunoreactivity,
but overall staining of lymphatics was weaker than blood vessels
( Fig. 3 A ). The distributions of PECAM-1 and VE-cadherin
partially colocalized at buttons at the borders of oak leaf – shaped
endothelial cells of initial lymphatics ( Fig. 3 B ) and at zippers
of collecting lymphatics ( Fig. 3 C ). At buttons, VE-cadherin
was located at the sides of fl aps, and PECAM-1 tended to have
a complementary distribution at the tip of fl aps ( Fig. 3, D – F ).
Arcs of PECAM-1 staining ~3 ? m in length varied from 0.5 to
2 ? m in width, depending on the amount of cell overlap. By
comparison, discontinuous segments of VE-cadherin staining
were ~3 ? m in length and ~0.5 ? m in width.
Contrasting properties of PECAM-1 and VE-cadherin
The complementary distributions of PECAM-1 and VE-
cadherin in initial lymphatics raised the possibility that the two
JEM VOL. 204, October 1, 2007
association with lymphatics, but after LPS, MHC II – positive
cells were more rounded and concentrated near lymphatics
( Fig. 6, A and B ). After LPS, 55% of initial lymphatics had clus-
ters of four or more MHC II – positive cells inside or within
10 ? m of their wall (411 lymphatics examined in fi ve tracheas).
Further measurements made after LPS exposure showed that
cell clusters were not uniformly distributed along button-rich
intranasal instillation of LPS. At this time, MHC II – positive
cells were abundant near lymphatics ( Fig. 6, A and B ) and
lymphatics were enlarged, with the mean diameter increased
21%, from 56 ± 1.5 ? m in pathogen-free mice to 68 ? 1.3
? m after LPS (P ? 0.05).
Under baseline condition, most MHC II – positive cells had
a dendritic phenotype ( Fig. 6 B , inset) and little or no apparent
Figure 2. Colocalization of VE-cadherin and tight junction proteins at buttons and zippers. (A – E) Confocal images showing button-like pattern
of VE-cadherin (left) paired with fi ve different tight junction – associated proteins (middle) at endothelial junctions of initial lymphatics. Corresponding
merged images (right) show that VE-cadherin colocalizes with all fi ve tight junction proteins in buttons (A – E). (F) Continuous, zipper-like distribution of
VE-cadherin (left) and occludin (middle) at endothelial junctions of collecting lymphatic; merged image (right) shows colocalization of the junctional
proteins in zippers. In each case, lymphatic vessel identity was determined by vascular endothelial growth factor receptor 3 immunoreactivity. LYVE-1 or
Prox1 were not used in these particular studies due to antibody species incompatibility issues. Bars: 10 ? m.
BUTTON-LIKE JUNCTIONS OF INITIAL LYMPHATICS | Baluk et al.
(430 ? m; Fig. 1 F ), and both were signifi cantly less than
the median overall length of mucosal lymphatics in tracheas
(980 ? m; Fig. 6 C ).
Leukocyte migration through the lymphatic endothe-
lium was visualized by confocal microscopy ( Fig. 6 B ), and
the transendothelial or intraluminal location of cells was con-
fi rmed by isosurface rendering of confocal image stacks and
image rotation ( Fig. 6, D and E ; and Video 1, available at
regions of lymphatics but were preferentially found near
the tips. Comparison of the position of cell clusters inside
or near infl amed lymphatics with the length of each vessel,
using the vessel tip as a reference point, revealed that ? 50% of
cell clusters were located within the fi rst 16% of the vessel
length. The median distance for the location of cell clus-
ters (160 ? m; Fig. 6 C ) was signifi cantly less (P ? 0.05) than
the median length of the button-rich region of lymphatics
Figure 3. Different distributions of VE-cadherin and PECAM-1 in lymphatics. (A) Overview of PECAM-1 immunoreactivity of blood vessels
(arrows) and lymphatics (arrowheads) in whole mount of mouse trachea. (B and C) Although VE-cadherin (red) and PECAM-1 (green) are both present in
lymphatic endothelial cells, they do not have identical distributions in initial lymphatics (B) or collecting lymphatics (C) and colocalize only in scattered
regions (yellow). (D – F) VE-cadherin (red, arrowheads) and PECAM-1 (green, arrows) have largely complementary distributions at buttons in initial lym-
phatics. The amount of colocalization is limited (yellow; F). Bars: (A) 100 ? m; (B and C)10 ? m; (D – F) 5 ? m.
JEM VOL. 204, October 1, 2007
Figure 4. Contrasting effects of loss of PECAM-1 or VE-cadherin in lymphatics. (A – E) Normal-appearing endothelial junctions in initial lym-
phatic (arrow) and blood vessel (arrowhead) in PECAM-1 – null mice. (A – C) Normal distribution of VE-cadherin immunoreactivity at buttons in initial
lymphatic and at zippers in blood vessel in PECAM-1 – null mouse. Lymphatic is marked by LYVE-1 immunoreactivity (green). (D and E) Normal distribu-
tion of VE-cadherin at buttons despite absence of PECAM-1 immunoreactivity in a PECAM-1 – null mouse (D) compared with complementary distribu-
tions of VE-cadherin and PECAM-1 in a wild-type mouse (E). (F – I) Disorganization of endothelial junctions in lymphatics and blood vessels 7 h after
inhibition of VE-cadherin by function-blocking BV13 antibody. (F and G) Normal distribution of VE-cadherin at buttons in initial lymphatic (arrow) and
at zippers in blood vessels (arrowheads) in a mouse injected with control IgG compared with disorganization of VE-cadherin and PECAM-1 immuno-
reactivities in initial lymphatic (arrow) and blood vessel (arrowhead) 7 h after injection of BV13 antibody (G). (H and I) Colocalization of VE-cadherin and
ZO-1 at normal buttons after control IgG (H) compared with dispersion of VE-cadherin, but not ZO-1, at buttons 7 h after BV13 antibody (I).
Bars: (A – C, F, G) 20 ? m; (D, E, H, I) 5 ? m.
BUTTON-LIKE JUNCTIONS OF INITIAL LYMPHATICS | Baluk et al.
through buttons. We attempted to address this issue by confocal
microscopic examination of lymphatics 24 h after M. pulmonis
infection, when leukocyte migration was especially abundant.
Although leukocytes clearly migrated through the endothelium
of button-rich regions of lymphatics ( Fig. 6, H and I ), they
obscured or distorted the junction at the site of transmigration,
and the precise relationship between migrating leukocytes and
buttons was ambiguous.
This study sought to defi ne the structural organization and
composition of endothelial junctions at sites in lymphatics
PECAM-1 – null mice exposed to LPS, leukocytes had the usual
association with the proximal part of initial lymphatics ( Fig.
6 F ), indicating that PECAM-1 was not essential for leuko-
cyte attraction to or migration into lymphatics. These fi nd-
ings suggest that migrating leukocytes preferentially entered the
proximal half of the segment of lymphatics with buttons at the
border of oak leaf – shaped endothelial cells.
Migration of leukocytes through endothelial junctions of
lymphatics was confi rmed by transmission electron micro-
scopic examination ( Fig. 6 G ), but imaging of thin (two-
dimensional) sections did not resolve whether cells migrated
Figure 5. Zippers at growing tips of lymphatic sprouts. (A – D) Confocal images of tracheal mucosa after M. pulmonis infection showing lym-
phatic sprouts in regions that do not contain lymphatics in pathogen-free mice. (A and B) Continuous VE-cadherin – positive zippers (arrows) at grow-
ing tips of lymphatic sprouts at 14 d after M. pulmonis infection compared with discontinuous buttons in the remainder of initial lymphatics
(arrowheads). Tips of lymphatic sprouts have little or no LYVE-1 immunoreactivity. (C and D) Most lymphatics identifi ed by Prox1 immunoreactivity
(arrows; C) have buttons (arrows; D) at 7 wk after infection. Some leukocytes have PECAM-1 immunoreactivity (arrowheads; D). Blood vessels have
strong VE-cadherin and PECAM-1 immunoreactivities (yellow; D). Boxed regions in A and C are enlarged in B and D, respectively. Bars: (A and C)
100 ? m; (B and D) 50 ? m.
JEM VOL. 204, October 1, 2007
Figure 6. Sites of leukocyte entry into initial lymphatics in airway infl ammation. (A) Whole mount of mouse trachea 24 h after intratracheal LPS.
MHC II – positive cell clusters (arrows, red) in or near initial lymphatics (green). (B) Enlargement of boxed region in A. Cells inside lymphatic (arrows) are
more rounded than dendritic cells in trachea of pathogen-free mouse (inset). (C) Distribution of MHC II – positive cell clusters along the length of tracheal
lymphatics, with the tip used as a reference. Half of the cell clusters were within 160 ? m of the tip. Values are presented as means ? SEM. (D and E)
Isosurface renderings of confocal images of MHC II cells (arrows) entering an initial lymphatic with buttons. (E) Enlargement of boxed region in (D).
(F) MHC II – positive cells near and inside initial lymphatic of a PECAM-1 – null mouse 24 h after LPS. (G) Transmission electron microscopic image of a leuko-
cyte (pink) migrating through an intercellular junction in endothelium (green) of tracheal lymphatic with prominent junctional fl ap (arrow; M. pulmonis
infection, 6 wk). (H and I) Confocal image (H) and isosurface rendering (I) of CD45-positive leukocytes (red, arrows) inside initial lymphatic 24 h after
infection by M. pulmonis . Endothelial cell junctions are marked by VE-cadherin (red). PECAM-1, green; LYVE-1, blue. See also Video 1, available at
http://www.jem.org/cgi/content/full/jem.20062596/DC1. Bars: (A) 200 ? m; (B) 50 ? m; (D) 10 ? m; (E) 5 ? m; (F) 50 ? m; (G) 2 ? m; (H and I) 20 ? m.
BUTTON-LIKE JUNCTIONS OF INITIAL LYMPHATICS | Baluk et al.
the endothelium is maintained. The results of our studies off er
a possible explanation ( Fig. 7 ). Openings between buttons are
ready candidates for the valves, and buttons at the sides of fl aps
would serve as anchors. Such specialized junctions that secure
the sides of interdigitating fl aps would permit fl uid entry
through openings at the tips of fl aps without repetitive dis-
assembly and reformation of endothelial junctions.
To our knowledge, intercellular junctions with the orga-
nization of buttons have not been previously described. The
button-rich nature of initial lymphatics and abrupt transition
to exclusively zippers in collecting lymphatics is consistent
with functional diff erences between regions specialized for
fl uid uptake and regions specialized for lymph transport.
The location and mechanism of plasma extravasation from
blood vessels has been extensively examined ( 33-37 ). Under
baseline conditions, tight junctions between endothelial cells
are crucial to normal barrier function ( 24, 38 ). In infl am-
mation, formation of focal intercellular gaps leads to plasma
leakage from venules ( 33, 39, 40 ). These gaps are structurally
dissimilar to buttons in lymphatics.
Proof that buttons anchor the borders of openings for
fl uid entry into initial lymphatics is lacking, in part because
where fl uid and cells enter. Endothelial cells of initial lym-
phatics were found to be interconnected by discontinuous
buttons. By comparison, collecting lymphatics downstream
had continuous zippers at cell borders without openings.
Buttons in the endothelium of initial lymphatics
Identifi cation of molecules involved in lymphatic specifi ca-
tion, development, and pathology, through the use of mo-
lecular tools and novel animal models, has greatly advanced
the understanding of the mechanism of lymphedema, im-
mune cell traffi cking, and tumor metastasis via lymphatics
(Tammela, T., personal communication) ( 1-4, 31, 32 ). Mul-
tiple features of lymph and cell transport from tissues to lym-
phatics to lymph nodes have also been elucidated ( 1, 4, 7, 9 ),
but understanding of the cellular mechanisms of fl uid and cell
entry into lymphatics has not advanced as far.
The distinctive oak leaf – shaped endothelial cells of initial
lymphatics has been assumed to be related to fl uid entry ( 9, 20 ).
Flaps at loosely connected borders of these cells have been
interpreted as primary valves that permit unidirectional fl ow of
fl uid into lymphatics ( 9, 10 ). Loose connection of endothelial
cells would raise the question of how the structural integrity of
Figure 7. Buttons in initial lymphatics border sites of fl uid entry. (A) Schematic diagram showing distinctive, discontinuous buttons in endo-
thelium of initial lymphatics and continuous zippers in collecting lymphatics. Both types of junction consist of proteins typical of adherens junctions
and tight junctions. (B) More detailed view showing the oak leaf shape of endothelial cells (dashed lines) of initial lymphatics. Buttons (red) appear to be
oriented perpendicular to the cell border but are in fact parallel to the sides of fl aps. In contrast, most PECAM-1 expression is at the tips of fl aps. (C and D)
Enlarged views of buttons show that fl aps of adjacent oak leaf – shaped endothelial cells have complementary shapes with overlapping edges. Adherens
junctions and tight junctions at the sides of fl aps direct fl uid entry (arrows) to the junction-free region at the tip without repetitive disruption and refor-
mation of junctions.
JEM VOL. 204, October 1, 2007
junctions between vascular endothelial cells in vitro ( 50, 51 ).
These fi ndings fi t with the importance of VE-cadherin in the
organization of buttons in initial lymphatics, but the rele-
vance of PECAM-1 is still unclear.
Maturity of buttons in lymphatics
To address the question of whether the presence of buttons
is a feature of lymphatic immaturity instead of lymphatic spe-
cialization, we examined the junctions of newly formed lym-
phatics where sprouting was active and junctions may be
immature. In an established model of sustained infl ammation
accompanied by robust lymphangiogenesis ( 21 ), we found
that the growing tips of lymphatic sprouts had zippers, not
buttons. The presence of zippers at the tip of lymphatic
sprouts indicates that continuous junctions can form rapidly
and that buttons are not a feature of the most dynamic, im-
mature region of lymphatic sprouts. However, over time all
but the tips of new lymphatics had buttons largely like those
present at baseline, despite elaborate expansion of the overall
network of lymphatics in parallel with the increased fl ux of
fl uid and immune cells.
Sites of leukocyte entry in initial lymphatics
The presence of openings between junctions at the border
of oak leaf – shaped endothelial cells raises the question of
whether buttons are sites of cell entry. At fi rst glance, open-
ings between buttons would seem attractive routes for cell
entry. The dimensions of the openings (?3 ? m) fi ts with the
size of gaps where leukocytes migrate through the endothe-
lium of infl amed venules ( 52 ) and with the size of pores
(3 ? m) that leukocytes migrate through in chemotaxis experi-
ments in vitro ( 14 ). Although the presence of PECAM-1 at
the tip of fl aps is consistent with involvement in leukocyte
traffi cking ( 45 ), the apparently normal leukocyte migration
in PECAM-1 – null mice weighs against an essential role in
the C57BL/6 strain. As loss of PECAM-1 results in impaired
leukocyte effl ux from blood vessels in mouse strains other
than C57BL/6 after exposure to infl ammatory stimuli ( 45 ), a
role for PECAM-1 in the rate or effi ciency of migration into
lymphatics cannot be excluded without studies of other
The preferential association of leukocyte clusters with the
proximal half of the region of lymphatics with buttons was an
unexpected fi nding. The mismatch between regions of cell
clusters and the overall extent of buttons indicates that precise
sites of leukocyte entry are regulated by additional factors
such as chemokines or adhesion molecules ( 4, 14, 53 ).
Did leukocytes enter through openings between buttons
in the proximal half of the button-rich region of initial lym-
phatics? Our lack of success in answering this question may
be caused partly by the infrequency of catching leukocytes in
the act of migration and partly by the temporary deformation
of junctions as migrating cells pass through them. Similarly,
we cannot exclude that some cells follow a transcellular route
( 54 ). Certainly, the understanding of leukocyte migration
into lymphatic vessels is in its infancy compared with what is
sites of fl uid entry into lymphatics have not been visualized
directly. The rapid movement of tracers from the interstitium
into initial lymphatics and then to collecting lymphatics blurs
the spatial resolution needed for defi nitive localization of en-
try sites at the cellular level ( 7, 41, 42 ). Our preliminary ex-
periments using fl uorescent 25-nm microspheres to identify
these sites confi rmed this rapid movement (unpublished data).
Similar factors may complicate the interpretation of results
favoring the contribution of transcytotic transport to fl uid
entry in initial lymphatics ( 43 ).
Adherens and tight junction proteins at buttons
The fi nding that both VE-cadherin and tight junction –
associated proteins were present in buttons seemed at fi rst to
confl ict with the traditional view that lymphatic endothelium
must have loose, poorly developed, or no intercellular junc-
tions to permit entry of fl uid and cells ( 7, 8, 44 ). However,
the observation that both buttons and zippers have the same
repertoire of junctional proteins, including occludin, claudin-5,
ZO-1, ESAM, and JAM-A, indicates that the principal diff er-
ence between these junctions is their organization rather
than their composition. The presence of discontinuous but
otherwise conventional tight junctions at buttons is consis-
tent with their function as anchoring points along the sides of
fl aps and as borders for openings for fl uid passage without jun-
Importance of VE-cadherin at buttons
The consistent presence of PECAM-1 and VE-cadherin in
the endothelium of initial lymphatics led us to examine the
importance of each protein to the organization of buttons.
PECAM-1 is known to be expressed in lymphatic endothe-
lial cells from immunohistochemical observations in vivo ( 21 )
and in culture ( 14 ) and from gene profi ling studies ( 14-17 ).
PECAM-1 – null mice are viable and have a normal vas-
culature ( 27 ), but PECAM-1 may contribute to maintenance
of vascular endothelial integrity in disease. As examples,
PECAM-1 – null mice have reduced transmigration of leuko-
cytes ( 26, 27, 34, 45 ), increased bleeding times and vascular
leakage ( 46, 47 ), and greater susceptibility to endotoxic shock
( 48 ). However, genetic background may contribute to the
functional signifi cance of PECAM-1, as PECAM-1 blockade
or deletion predisposes FVB/n mice to chronic pulmonary
infl ammation and fi brosis but has little or no eff ect on the in-
fl ammatory response of C57BL/6 mice ( 45, 49 ). In our studies
of C57BL/6 PECAM-1 – null mice, we detected no change in
the distribution of VE-cadherin at buttons or in the integrity
of buttons in initial lymphatics. This fi nding is consistent with
the association of PECAM-1 with openings between intercel-
lular junctions instead of with the junctions themselves.
By comparison, inactivation of VE-cadherin at adherens
junctions, by administration of function-blocking antibody
BV13, resulted in dispersion of VE-cadherin at buttons and
zippers in lymphatics, as shown previously for junctions in
blood vessels ( 28 ). ZO-1 was not similarly aff ected. After
BV13, PECAM-1 was dispersed at buttons in vivo but not at
BUTTON-LIKE JUNCTIONS OF INITIAL LYMPHATICS | Baluk et al.
clonal AB5475; Chemicon), or vascular endothelial growth factor receptor 3
(goat polyclonal antibody AF743; R & D Systems). Adherens junction was
VE-cadherin (rat clone BV13 and rabbit polyclonal antibody). Tight junc-
tions were ESAM (rat clone 1G8.2), ZO-1 and occludin (rabbit polyclonal
anti bodies 40-2200 and 71-1500; Zymed Laboratories), and JAM-A and
claudin-5 (rat clone BV12 and rabbit polyclonal antibody E2.8, respectively).
PECAM-1 was CD31 (hamster anti – mouse PECAM-1, clone 2H8; Chemi-
con). Leukocytes were MHC II (rat clone M5/115.14.2; eBioscience) or
CD45 (rat clone Ly-5; BD Biosciences). Secondary antibodies were labeled
with FITC, Cy3, or Cy5 (Jackson ImmunoResearch Laboratories). Speci-
mens were viewed with a fl uorescence microscope (Axiophot; Carl Zeiss
MicroImaging, Inc.) with a 3CCD low light red-green-blue (RGB) video
camera (CoolCam; SciMeasure) or a confocal microscope (LSM-510; Carl
Zeiss MicroImaging, Inc.) using AIM confocal software (version 3.2.2).
Morphometric measurements. The vessel diameter and length, width,
and area of endothelial cells were measured in real-time images of lymphatics
and venules in tracheas stained for VE-cadherin and PECAM-1 by using a
digitizing tablet linked to a video camera on the Axiophot microscope with
40 ? (NA 1.0) or 63 ? (NA 1.4) objectives. Cell perimeter and shape factor
were measured as previously described ( 52 ). The total length of 411 mucosal
lymphatics and the distance from the tip of lymphatics to the location of 228
cell clusters, consisting of four or more MHC II – positive cells inside or
within 10 ? m of the wall of a lymphatic, were measured in fi ve tracheas 24 h
after intranasal instillation of LPS. Tracheas were stained for MHC II and
LYVE-1 immunoreactivities and imaged with 5 ? (NA 0.32) or 20 ? (NA
0.75) objectives. The distribution of 3,110 buttons was assessed along the
length of fi ve lymphatics in the trachea of each of fi ve pathogen-free mice.
Isosurface rendering of confocal images. Confocal RGB image stacks
were imported into Imaris software (version 5.0.3; Bitplane). Voxels with
fl uorescence intensities above a certain threshold were assigned for each
color channel. Isosurfaces were rendered from these voxels and smoothed
with a Gaussian fi lter, creating three-dimensional reconstructions in which
the spatial resolution was conserved.
Scanning and transmission electron microscopy (EM). For scanning
EM, tissues were fi xed by vascular perfusion of fi xative containing 2% glu-
taraldehyde in 100 mmol/liter of phosphate buff er, treated with 30% potas-
sium hydroxide at 60 ° C for 8 min to dissolve the extracellular matrix, stained
with 2% tannic acid and 1% OsO 4 , dehydrated with ethanol, critical point
dried, coated in an osmium plasma coater (OPC60A; Filgen), and examined
with a scanning electron microscope (S-5000; Hitachi) ( 22, 23 ). Transmis-
sion EM was performed as previously described ( 23, 52 ).
Statistical analysis. Values are presented as means ? SEM with four to fi ve
mice per group, unless otherwise indicated. The signifi cance of diff erences
between means was assessed by analysis of variance, followed by the Dunn-
Bonferroni test for multiple comparisons. P ? 0.05 was considered signifi cant.
The signifi cance of diff erences between distributions of buttons, clusters of
dendritic cells along the length of lymphatics, and lengths of lymphatics in
tracheal mucosa was analyzed by the Kolmogorov-Smirnov two-sample test.
Online supplemental material. Fig. S1 depicts confocal and scanning
electron microscopic images of junctions in initial and collecting lymphatics
in other organs. Video 1 shows three-dimensional aspects of leukocytes
interacting with lymphatic endothelial cell junctions. Online supple-
mental material is available at http://www.jem.org/cgi/content/full/jem
We thank Maria Grazia Lampugnani for assistance with experiments in Milan and
helpful discussions, DongJi Zhang for production of M. pulmonis stock, and Amy
Haskell for transmission EM.
This work was supported in part by grants from the National Heart, Lung,
and Blood Institute (HL-24136 and HL-59157) and the National Cancer Institute
known about leukocyte attachment and migration via inter-
cellular and transcellular routes through the endothelium of
venules ( 34-37, 55 ).
In conclusion, the borders of distinctive oak leaf – shaped
endothelial cells of initial lymphatics are joined by specialized
buttons ( Fig. 7 A ). The discontinuous feature of buttons dis-
tinguishes them from zippers in collecting lymphatics ( Fig.
7 A ), but both types of junctions are composed of proteins typ-
ical of adherens junctions and tight junctions found in the
endothelium of blood vessels. Buttons seal the sides of fl aps at
the border of oak leaf – shaped endothelial cells ( Fig. 7 B ),
leaving open the tips of fl aps as routes for fl uid entry without
disassembly and reformation of intercellular junctions ( Fig. 7,
C and D ). VE-cadherin is essential for maintaining the integ-
rity of buttons, but PECAM-1, though strategically located at
the tip of many fl aps, is not essential for button integrity or
leukocyte entry, at least not in C57BL/6 mice. Most leuko-
cytes enter the proximal half of button-rich regions of initial
lymphatics, but the exact site of entry in relation to buttons is
unresolved. Collectively, our fi ndings show that buttons are
likely sites for fl uid entry into initial lymphatics but are not
the sole determinants of leukocyte entry.
MATERIALS AND METHODS
Mice. Specifi c pathogen-free C57BL/6 mice (Charles River Laboratories) of
either sex were housed under barrier conditions. PECAM-1 – null mice on a
C57BL/6 background, as previously described ( 27 ), were originally donated
by T. Mak (Amgen Institute, Toronto, Canada). Mice were anesthetized by
intramuscular injection of 100 mg/kg ketamine and 10 mg/kg xylazine. All
experimental procedures were approved by the Institutional Animal Care
and Use Committees of the University of California, San Francisco (UCSF)
and the FIRC Institute of Molecular Oncology Foundation. All reagents
were purchased from Sigma-Aldrich unless indicated otherwise.
Mouse models of infl ammation. Infl ammation was induced by intranasal
inoculation of mice with 250 ? g LPS (type 055:B5) in 50 ? l PBS ( 56 ) or
50 ? l of broth containing 10 6 CFU of M. pulmonis organisms (strain CT8),
as previously described ( 21 ). Mice were anesthetized before inoculation and
allowed to recover. At 24 h after LPS or 24 h to 40 d after M. pulmonis
inoculation, mice were anesthetized again for further studies. M. pulmonis
organisms activate an immune response with a time course similar to other
airway infections ( 57, 58 ). Robust lymphangiogenesis begins about 7 d after
infection ( 21 ).
In vivo blockade of VE-cadherin. C57BL/6 mice were injected via the
tail vein with 100 ? g BV13, a function-blocking rat monoclonal anti – mouse
VE-cadherin antibody or with a control rat IgG ( 28 ). Tracheas were exam-
ined 3 or 7 h later. LPS-induced infl ammation was not studied in mice after
VE-cadherin inhibition by BV13 because of the rapid induction of progressive
plasma leakage, interstitial edema, hemorrhage, and death within 24 h ( 28 ).
Immunohistochemistry. Mice were perfused for 2 min with fi xative (1%
paraformaldehyde in PBS, pH 7.4) ( 21 ) from a cannula inserted through the
left ventricle into the aorta. The trachea, diaphragm, urinary bladder, ear,
and tail skin were removed and immersed in fi xative for 1 h at 4 ° C. Tis-
sues were washed and stained immunohistochemically by incubating whole
mounts with one or more primary antibodies diluted in PBS containing 0.3%
Triton X-100, 0.2% bovine serum albumin, 5% normal goat serum, and
0.1% sodium azide, as previously described ( 21 ). Lymphatics were LYVE-1
(rabbit polyclonal 07-538; Upstate Biotechnology), Prox1 (rabbit poly-
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(CA-82923) of the National Institutes of Health (to D. McDonald), by a grant from the
Deutsche Forschungsgemeinschaft (SFB492; to D. Vestweber), and by a grant from
the Associazione Italiana per la Ricerca sul Cancro and the European Community
(lymphangiogenomics project grant LSHG-CT-2004-503573; to E. Dejana). J. Fuxe
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Union Against Cancer American Cancer Society International Fellowship for
The authors have no confl icting fi nancial interests.
Submitted: 12 December 2006
Accepted: 15 August 2007
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